We examined environmental smooth and rough V. cholerae isolates (RGVCs) collected at two locations along the Rio Grande to study T6SS regulation in V. cholerae exposed to microbial competitors and predators.
Our study showed that smooth RGVC isolates use their T6SS to kill other Gram-negative bacteria isolated from the Rio Grande delta. Deletion of the T6SS gene vasK resulted in a loss of bacterial killing. Importantly, the killing phenotype was restored by vasK complementation in trans. The requirement of VasK for killing implies that a constitutively active T6SS provides smooth RGVC isolates with a competitive advantage compared to their bacterial neighbors. By killing other bacteria, RGVC isolates might enhance their own survival in their environmental niche. In addition, we found that V. cholerae isolates use their T6SS to compete against each other.
In our experiments, Hcp synthesis and secretion correlated with eukaryotic and prokaryotic host cell killing (). For example, smooth Hcp-secreting RGVC isolates DL4211 and DL4215 () displayed full virulence towards E. coli
() and D. discoideum
(). Rough RGVC isolates with their frameshift mutations in the T6SS transcriptional activator gene vasH
did not produce or secrete Hcp, and their virulence was attenuated. Sequencing and gene alignments of the T6SS transcriptional activator vasH
in rough strains indicated a missing guanine at position 157 in rough isolates, resulting in a frameshift mutation. Because VasH was recently implicated in regulating both the large and auxiliary T6SS gene clusters in V. cholerae
, we speculated that the vasH
frameshift mutation in the rough isolates silences T6SS expression. However, trans
-complementation of the vasH
mutation by episomal expression of V52′s vasH
restored synthesis but not secretion of the T6SS hallmark protein Hcp (). Trans
-complementation with vasH
from N16961, which is more closely related to vasH
from RGVC isolates DL2111 and DL2112 (), restores Hcp synthesis and secretion in a vasH
mutant of V52, but only restores Hcp synthesis (and not secretion) in a vasH
mutant of N16961 
. Thus, we believe that the inability to restore Hcp secretion in rough strains is not a reflection of the polymorphic nature of VasH.
Secretion and virulence phenotypes of RGVC isolates.
At this time, it is unclear whether selective pressures for T6SS regulation exist that drive constitutive T6SS expression in smooth isolates and disable T6SSs in rough V. cholerae
strains. V. cholerae
LPS's O-antigen has been shown to induce protective immune responses in humans and experimental animals 
. To counteract the host immune response, V. cholerae
may use its T6SS to kill phagocytic immune cells such as macrophages 
. Because rough isolates lacking O-antigen are frequently isolated from convalescent cholera patients 
, repression of O-antigen biosynthesis may represent an immune evasion mechanism for V. cholerae
. Such evasion would allow the pathogen to persist in the host, perhaps in a subclinical state as rough V. cholerae
have been shown to be avirulent. In this scenario, rough V. cholerae
does not require a functional T6SS, but tolerates mutations that disable its expression. Rough isolates have been shown to revert to a smooth, virulent state 
but it remains to be determined whether newly reverted smooth bacteria restore expression of their disabled T6SSs. We did not observe restoration of the T6SS in rough isolates through uptake and homologous recombination of chromosomal DNA from a T6SS+
donor, because rough isolates remained T6SS-negative in the presence of smooth T6SS+
strain V52 (data not shown).
El Tor strains possess a tightly controlled T6SS 
and thus differ from the smooth RGVCs that express the T6SS constitutively. As pandemic strains are believed to originate from environmental strains, we speculate that constitutive T6SS expression is prevalent in V. cholerae
exposed to microbial competitors and predators until virulence factors such as cholera toxin and toxin-coregulated pilus genes are acquired. However, how pandemic V. cholerae
regulate expression of T6SS during their complex life cycle remains to be determined.
It is becoming increasingly clear from our investigation and other reports 
that T6SS-expressing V. cholerae
deploy bactericidal effector proteins. Therefore, T6SS expression is likely tied to a protective mechanism, a form of T6SS-immunity that prevents the effector proteins from harming bacteria within a clonal population. We postulate that V52, DL4211, and DL4215 employ unique sets of toxin/antitoxin gene products and therefore form distinct compatibility groups. Members of a T6SS compatibility group could coexist because they encode antitoxins that match the cognate toxins. Conversely, members of different T6SS compatibility groups kill each other since the antitoxins of one compatibility group do not protect against the toxins of the other group. Hence, T6SS-mediated selective interstrain killing allows V. cholerae
to distinguish self from nonself. This form of kin selection may permit the evolution of distinct lineages, including those that give rise to toxigenic strains. The observations presented in this study indicate that the T6SS contributes to V. cholerae
's pathogenesis and fitness by providing an advantage in interspecific competition with eukaryotes or prokaryotes, and intraspecific competition with V. cholerae